An immunoadjuvant saponin fraction from Quillaja brasiliensis leaves was investigated by direct infusion and liquid chromatography/electrospray ionization ion trap multiple-stage mass spectrometry in negative ion mode (DI-ESI-IT-MSn and LC-ESI-IT-MSn ). The aglycone and the sequence of the oligosaccharide residues at C-3 and C- 28 were characterized based on MS2 and MS3 experiments of the [Me-H]- ions. According to their [Me-H]- ions, characteristic product ions and retention times, 27 bidesmosidic saponins, bearing four types of triterpenic aglycones, were tentatively identified.